Wiskott–Aldrich syndrome: Oral findings and microbiota in children and review of the literature

Abstract Objective Wiskott–Aldrich syndrome (WAS) is a rare X‐linked primary immunodeficiency, characterized by micro‐thrombocytopenia, recurrent infections, and eczema. This study aims to describe common oral manifestations and evaluate oral microbioma of WAS patients. Material and methods In this cohort study, 11 male WAS patients and 16 male healthy controls were evaluated in our Center between 2010 and 2018. Data about clinical history, oral examination, Gingival Index (GI) and Plaque Index (PI) were collected from both groups. Periodontal microbiological flora was evaluated on samples of the gingival sulcus. Results WAS subjects presented with premature loss of deciduous and permanent teeth, inclusions, eruption disturbance, and significantly worse GI and PI. They also showed a trend toward a higher total bacterial load. Fusobacterium nucleatum, reported to contribute to periodontitis onset, was the most prevalent bacteria, together with Porphyromonas gingivalis and Tannerella forsythia. Conclusions Our data suggest that WAS patients are at greater risk of alterations in the oral cavity. The statistically higher incidence of periodontitis and the trend to higher prevalence of potentially pathological bacterial species in our small cohort, that should be confirmed in future in a larger population, underline the importance of dentistry monitoring as part of the multidisciplinary management of WAS patients.


| INTRODUCTION
Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency, characterized by microthrombocytopenia, eczema, recurrent infections, and an increased incidence of autoimmunity and malignancies, with an incidence of $1-10 per 1 million individuals (Aldrich et al., 1954;Ochs & Thrasher, 2006;Orange et al., 2004). The median age at diagnosis in patients without a family history is 24 months (Buchbinder et al., 2014). Advances in the diagnosis and the availability of definitive treatments have significantly improved the prognosis of WAS patients (Aiuti et al., 2013;Brigida et al., 2016;Ferrua et al., 2019;Marangoni et al., 2009;Moratto et al., 2011), while the morbidity and the quality of life of untreated patients is still poor (Massaad et al., 2013).
Both in pediatric and adult patients, WAS is typically associated with gingivitis and periodontitis; moreover, petechiae in the oral mucosa and active bleeding may also be observed (Kita et al., 2013;Lee et al., 2007;Reddy & Binnal, 2011;Salazar et al., 2013;Sollecito et al., 2005;Szczawinska-Poplonyk et al., 2009). However, until now, few studies, mainly case reports, have addressed dental and oral manifestation in WAS patients, and the implications for oral health are yet to be clearly defined (Kita et al., 2013;Lee et al., 2007;Reddy & Binnal, 2011;Salazar et al., 2013).
The first aim of this study is to describe common oral signs and symptoms of individuals affected by WAS. The second aim is to evaluate the microbiota of the oral cavity in WAS patients, focusing mainly on the main pathogenic periodontal bacteria. The results emerging from these analyses may lead to early detection of oral disease in WAS patients, possibly helping to establish prevention measures to improve dental health in this patients' population.

| Patients
A total of 11 patients from an international cohort (all males, median age: 10 years, range 6-14), with WAS were evaluated at Pediatric All patients presented with a severe Zhu score (> or =3), as a result of different mutations in WAS gene (Zhu et al., 1995). Four of these patients presented autoimmune/autoinflammatory manifestations (namely vasculitis, inflammatory bowel disease, pyoderma gangrenosum) and two were on treatment with immunosuppressants as steroids plus IL-1RA and mesalazine, respectively (Brigida et al., 2016;Ferrua et al., 2019), at the time of oral sampling and microbiota evaluation. These four patients also were among the patients with the worst Zhu clinical score. None of the patients had assumed antibiotic therapy or prophylaxis in the 3 months prior to samples collection. A control group of 16 healthy male patients (median age: 10 years, range 8-12) was analyzed in parallel at the Unit of Dentistry, Division of Orthodontics of San Raffaele Hospital (Milan, Italy) between 2010 and 2018. Controls were matched for age and sex, and had to fulfill the following criteria: (1) no smoking; (2) no known systemic disease; (3) no alveolar bone loss visible on X-ray; (4) no fixed restorations or removal dentures; (5) no use of antibiotics within 3 months before the study; (6) no periodontal therapy within the previous 6 months.
Before the study, all participants received a standardized oral hygiene instruction by the same dental specialist and had a good oral hygiene profile.
Collection of biological specimen from patients was performed after parents/patients' signature of informed consent for biological samples collection, in the context of a protocol approved by the Ethi-

| Diagnostic examination
Clinical records were obtained, with particular focus on oral dental anamnesis for both groups. The routine oral and dental evaluation consisted of oral clinical examinations, intra and extra-oral photographs, lateral cephalometric, and panoramic radiographs.
The following parameters were recorded from the clinical history (presence/absence): gingival bleeding, gingivitis, periodontitis, caries, oral abscesses, aphthous lesions, early deciduous teeth loss, early permanent teeth loss, permanent teeth inclusions, and eruption disturbances. We also excluded the presence of other factors that could alter oral flora, such as diabetes, gastroesophageal reflux, use of orthodontic devices (Lucchese et al., 2018).
Furthermore, Gingival Index (GI) and Plaque Index (PI) were used to measure bleeding and periodontal symptoms (Löe, 1967). Specifically, the GI was used for the assessment of the gingival condition and records changes in the gingiva. It scores the marginal and interproximal tissues separately on a scale from 0 to 3 (Löe, 1967). The scores of the four areas of the tooth were added and divided by four to give the GI for the tooth.
The PI is used together with GI, and in our study preceded the gingival examination (Löe, 1967). It is used on all the erupted teeth.
There is no substitution for any missing tooth. It is used on all surfaces (mesial, occlusal, distal, lingual). This index measures the thickness of plaque on the gingival third. The PI of the patient was obtained by adding the values of each tooth and dividing by the number of teeth examined. The PI was scored for all teeth surfaces. Data collection was performed by a professional/skilled operator.

| Sample collection, DNA extraction, and quantification
After the removal of supra-gingival biofilm, a sample of the gingival sulcus microbiota was taken from a single site using sterile paper probes to detect the presence of the main six periodontal bacteria (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Fusobacterium nucleatum, Campylobacter rectus) (Bale et al., 2017;Lagier & Threadgill, 2014).
The site of the analysis was the second superior left deciduous molar or the first-second superior left permanent premolar.

| Methods error
The intraexaminer reproducibility was assessed after the examiner was trained according to the calibrated professional's criteria. Three healthy subjects of the control group were randomly selected and recruited for the calibration of GI and PI (Löe, 1967). The evaluation of Cohen's Kappa coefficient was used to assess intraexaminer agreement for the dichotomous variables, which have only two levels, that is, presence or absence of caries (Cohen, 1960;McHugh, 2012). Conversely, to quantify the measurement error of the variables, such as GI and PI, the formula described by Dahlberg was used (Dahlberg, 1940).
Five patients were randomly selected and re-observed by one operator; the scores (GI and PI) were reassigned twice by the same observer within 20 min of the first assessment (Dahlberg, 1940;Galvao et al., 2012). Intraexaminer error was <5% (95% confidence).

| Statistical analysis
Statistical descriptive analysis of the oral data and of medical signs and symptoms recorded was performed using IBM SPSS Statistics 21 software. The statistical analysis of microbiological characterization was performed on relative quantity calculated as ratios between the amount of each bacterial species (absolute quantity) and the total quantity of the bacteria. This allowed the reduction of variability due to random factors such as specimen preservation, DNA extraction efficiency and purification yield, as well as variability due to systematic factors (i.e., the higher amount of bacteria expected in specimens from deeper pockets characterizing oral inflammation) (Scapoli et al., 2015). To compare the different general health conditions between the two groups, the Fisher's exact test was applied; twotailed t test was applied to compare clinical parameters and bacterial species in healthy controls and WAS patients. Pearson correlation was used to measure the correlation between Zhu score and GI or PI.

| General health status
All the patients analyzed presented with classical WAS, characterized by a history of recurrent infections, eczema, thrombocytopenia, and hemorrhagic diathesis (associated to major bleeding events in the 54.6% of patients), showing a health-related quality of life (HRQOL) of 75.0 ± 10.0 (Selewski et al., 2015). In addition, 63.6% of these patients presented with anemia ( Figure 1). These disease-specific clinical features were confirmed to be significantly different in WAS patients as compared to the control group of healthy patients ( Figure 1).

| Oral health status
The major oral findings detected in WAS patients were: oral petechiae, gingivitis, and gingival bleeding in all of them, followed by aphthous lesions (72.7%), periodontitis (54.6%), and severe oral infections such as caries (63.6%) and oral abscesses (45.5%) (Figure 1). No other enamel abnormalities were detected. In comparison with age matched healthy subjects, the incidence of gingival bleeding, gingivitis, periodontitis, oral petechiae, and aphthous lesions was significantly higher in WAS patients (Figures 1 and 2a,b). Regarding orthodontic malocclusions in WAS patients, oral examination, and orthopantomographies revealed premature loss of deciduous (36.36%) and permanent (18.18%) teeth, inclusions (9.1%), and eruption disturbance (36.36%) (Figures 1 and 2c Table 1. GI and PI resulted worse in patients as compared to controls (Table 1). GI and PI were more severe in some of the patients with a higher Zhu score, despite the lack of a statistically significant correlation (p value 0.47 and 0.92, respectively).

| Oral microbiota
The absolute and the relative amount of each analyzed bacterium were retrieved in the samples of the 11 WAS patients and in the control group, in addition to the total charge of crevicular bacteria.
Despite the limitation given by the small sample size, due to the rarity of the disease, the oral microbiota samples revealed an almost 30% higher bacterial load in WAS patients, including species strongly associated with periodontitis (Bale et al., 2017;Lagier & Threadgill, 2014), if compared to age matched healthy donors. In particular, we detected a high to very high bacterial load for P. gingivalis (bacterial load: absolute amount 629, relative amount 0.85%), and T. forsythia (bacterial load: absolute amount 650, relative amount 0.82%). F. nucleatum, reported to contribute to the onset of periodontitis, was the most prevalent bacteria detected, with a very high bacterial load (bacterial load: absolute amount 1484, relative amount 1.88%) ( Table 2) (Hartsfield, 1994).
In WAS patients, a trend toward a higher total bacterial load in comparison to the control group was observed (p = 0.0576), while no significant differences were seen between the bacterial species analyzed in the two groups (Table 2).
Our study has shown that an increase in the main periodontal parameters (GI and PI) is observed in patients with WAS, highlighting that in these patients there may be a greater risk of oral disease. In  Note: Absolute amount: <100 is equal to "moderate" bacterial load; <500 means "high" bacterial load and >500 corresponds to "very high" bacterial load. Relative amount: <0.1000 is equal to "moderate" relative charge; <0.5000 is equal to "high" relative charge; <1.0000 corresponds to "very high" relative charge. Abbreviation: WAS, Wiskott-Aldrich syndrome; CTR, Control.  Despite the knowledge collected in the last few decades, many questions about oral pathophysiology in WAS patients remain unanswered. Hartsfield proposed WAS in itself as a cause of premature exfoliation of deciduous teeth and argued that the immunodeficiency could result also in early loss of permanent teeth (Hartsfield, 1994).

T A B L E 3 Review of the literature
To date, it is still unknown if these manifestations in WAS patients could be related to WASP defect in the osteoclasts compartment, that might determine their altered cellular function. Indeed, WASPdeficient mice presents defects in bone reabsorption due to the critical role of WASP in actin reorganization and podosome formation (Calle et al., 2004;Chellaiah et al., 2007). On the other hand, these manifestations are likely to be related to untreated inflammatory and infectious processes, which can give rise to early loss of deciduous teeth, delay in the eruption of the permanent teeth, disruption in the physiological sequence of teeth eruption and malocclusions, leading to further orthodontic problems.
It is widely reported that oral infections seem to increase the risk of cardiovascular disease and pulmonary disease (Bale et al., 2017;Li et al., 2000). The impact could be even higher in case of a primary immunodeficiency, therefore a more accurate prevention and treatment plan of the oral deterioration is mandatory in WAS patients to reduce local inflammation, bacteremia, and to prevent systemic complications.
It is known that orthodontic treatments can cause a higher amount of dental plaque and a higher risk of enamel and dentin alterations, with the possible appearance of white spot lesions and dental caries (Lucchese et al., 2018;Prati et al., 1999;Lucchese et al., 2011), even in healthy subjects. Again, these corrective measures could be even more critical in WAS patients, who are more susceptible to dental infections. Therefore, it is mandatory to increase awareness of this entity and to include a dental specialist evaluation in the multidisciplinary management of these patients in order to specifically address oral cavity alterations.
This is the first report addressing findings of oral manifestations and microbiota in a cohort of 11 WAS patients, with comparison to healthy subjects. Future studies on a larger WAS population are needed to add new details and to go deeper in the understanding of the factors that may underlie the pathogenesis of oral manifestations in WAS patients. We also think that the extension of the sampling to patients' family members in a prospective fashion and the study of a larger number of oral species could be interesting. Moreover, it will be interesting to observe how these findings may change after definitive treatments as HSCT or GT.

ACKNOWLEDGMENTS
We thank all local referring doctors who helped with patients' management; and all patients who participated in this study and their families.

CONFLICT OF INTEREST
The authors declare no conflicts of interests.

AUTHOR CONTRIBUTIONS
Alessandra Lucchese contributed to the conception, data acquisition, data interpretation, and wrote the manuscript. Sabina Cenciarelli contributed to the writing of the paper, data acquisition and data analysis. Pia Cicalese contributed to conception, design, data acquisition, analysis, and interpretation, and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.